mechanisms. Therefore, lipotypes are asso-
ciated with cell states, yet cell states are not
endowed with transcriptional programs that
would account for the lipotypes with which
they are associated. This raises the question
of whether lipotypes are causally upstream of
cell states and if lipid composition influences
cell-to-cell transcriptional heterogeneity.
To test this hypothesis, we treated dHFs
with the Cer synthase inhibitor FB1, which
blocks the production of sphingolipids (figs.
S1F and S2B), and performed scRNA-seq.
When FB1-treated dHFs were integrated in
the same transcriptional embedding along
with control cells, they displayed a different
distribution across states (Fig. 6B and fig.
S8C). FB1-treated cells were more frequently
associated with fibrolytic (from 6% in control
cells to 23% in FB1-treated cells) and vascu-
lar (from 0.6 to 1.3%) than fibrogenic (from
48 to 40%) and inflammatory (from 9 to 6%;
Fig. 6B and fig. S8D) states. These changes
correspond to an increased association of FB1-
treated dHFs with“papillary”and decreased
association with“reticular”fibroblast states
(Fig. 6C).
FB1 treatment deprives cells of most sphin-
golipids ( 43 ), so this treatment does not inform
on how the individual lipid species associ-
ated with the cell states influence signaling.
We established dHF lines overexpressing either
GM3S or Gb4S, two enzymes driving alterna-
tive sphingolipid-processing pathways (Fig.
6D). These overexpressing (OE) cells dis-
played the expected changes in sphingolipid
composition, with GM3S-OE dHFs composedlargely of ChTxB+cells and Gb4S-OE dHFs
composed largely of ShTxB1a+/2e+cells (Fig. 6E
and fig. S8E).
GM3S-OE and Gb4S-OE lines were then
analyzed by scRNA-seq to test the impact of
lipotype change on cell state. GM3S-OE and
Gb4S-OE dHFs populated two distinct tran-
scriptional regions (Fig. 6F). Gb4S-OE dHFs
were more associated with basal, inflamma-
tory, and fibrolytic states, whereas GM3S-OE
dHFs were for the major part in a fibrogenic
state (88%) and almost never in inflammatory
or fibrolytic states (fig. S8F). Gene expression
analysis confirmed this transition:COL12A1
andVCANmarkers of fibrogenic state were
significantly up-regulated in GM3S-OE cells
and down-regulated in Gb4S-OE cells, whereas
the fibrolytic and inflammatory markersMMP-1Capolupoet al.,Science 376 , eabh1623 (2022) 15 April 2022 8 of 12
CTRLFB1
TreatmentBCp-Lenti Gb4S-V5-OE GM3S-V5-OEShTxB2eShTxB1aC
hTxBE
D F
p-Lenti Gb4S-V5-OE GM3S-V5-OEanti-V5anti-GOLPH3p-Lenti Gb4S-V5-OE GM3S-V5-OE0.75- 0.75
Avg.Z-scor
eClusters Cell densityUMAP2UMAP1UMAP2UMAP1yti
sn
ed(^) g
ol
-4
-5
-6
ytis
ne
d (^) g
ol
-4
-5
-6
FB1 Treatment
Papillary
signatures
Reticular
signatures
p-Lenti Gb4S-V5-OE GM3S-V5-OE
Papillarysignatures
Reticularsignatures
G
Toxin staining (intensity)
Enzyme expression (spots cells)
ST3GAL5
(^05)
15
ChTxB
R = 0.03
A4GALT
(^03)
50
ShTxB1a
R = 0.29
ChTxB
ST3GAL5
A4GALT
ShTxB1a
A
0.75
- 0.75
Avg. Z-scoreFig. 6. Effect of sphingolipid perturbations on cell states.(A) Representative
confocal micrographs of correlative mRNA-FISH/fluorescence toxin staining
usingA4GALTandST3GAL5(magenta) probes and ShTxB1a (green) and ChTxB
(blue). Nuclei were labeled with Hoechst (gray). Scale bar, 50mm. Right,
scatterplot showing the level of expression against toxin fluorescence. PearsonÕs
correlation coefficient is indicated. Quantification on 120 and 96 individual
cells forA4GALTandST3GAL5, respectively. (B) Left, UMAP embedding of the
scRNA-seq data for the control (5652 individual dHFs) and FB1-treated cells
(6546 individual dHFs). Cells are colored by their assigned cluster. Right, density
maps of control (CTRL) and FB1-treated cells mapped in the UMAP space.
(C) Papillary and reticular gene signatures in the FB1-treated sample overlaid
onto the UMAP embedding. (D) Confocal images of the overexpressing cell
lines stained with antibodies against V5 protein tag (green) and GOLPH3 (red).
Scale bar, 50mm. (E) Confocal images of the overexpressing cell lines stained
with the bacterial toxins ShTxB1a (red), ShTxB2e (green), and ChTxB (blue).
Scale bar, 50mm. (F) Cell density plot of single-cell expression profile of the OE
dHF cells mapped by similarity onto the UMAP projection in (B). (G) Papillary
and reticular signatures overlaid onto the UMAP embedding of the OE dHFs.RESEARCH | RESEARCH ARTICLE